Transporters for Glucocorticoids: Exploring a New Paradigm for Steroid Hormone Regulation and a Potential Strategy for Identification of Toxin/Disruptor Transporter Machineries A well-entrenched paradigm holds that steroid hormones, like glucocorticoids (GCs), diffuse freely across plasma membranes in order to access their intracellular receptors and influence gene transcription. This view persists despite biochemical, genetic, and cell biological evidence, albeit sporadic, consistent with mediated transport (herein referred to as any process that moves molecules across plasma membranes, including active transport, endocytosis/pinocytosis, passage through pores or channels, and/or coupling to carrier proteins) of steroids. Nearly two decades ago, for example, we identified a conserved ATP-binding-cassette transporter that selectively exports dex in yeast, and showed that a drug that inhibits the yeast activity also leads to increased intracellular dex in mammalian cells. Nevertheless, the widely held assumption that steroids diffuse passively through membranes precluded a focused inquiry into possible transporters. Here, we propose a tripartite project to identify steroid transporters. A distinctive element of our strategy is its sensitivity, achieved either by specific binding of intracellular GCs to the glucocorticoid receptor (GR), which in turn stimulates increased transcription and translation of fluorescent reporter proteins, or by covalent affinity labeling of steroid-interacting proteins and identification by ultrasensitie mass spectrometry. Our specific approaches are (i) to identify candidate machineries for GC transport via reverse and forward genetics, (ii) to identify GC-interacting proteins via chemistry and proteomics, and (iii) to test and validate roles for candidate proteins in GC transport via targeted genome editing in mice. Success of this exploratory project would provoke future research extending in at least two major directions. First, machineries that specify the transit of steroids across plasma membranes will likely participate in novel gene regulatory mechanisms, uncover new avenues to cell specificity of hormone action, and contribute to both the cause and the understanding of endocrine diseases. Second, identification of transport machinery for steroids will motivate re-evaluation of cellular access for other lipophilic small molecules - physiologic, pathologic and pharmacologic, as well as environmental toxins. Indeed, transporters for other such molecules are likely to reside in the same or related gene families as those discovered for glucocorticoids. A simple re- execution of our GC-based strategy using a different hormone or receptor-dependent toxin/endocrine disruptor (for example, estradiol, an environmental estrogen, or an aromatic hydrocarbon) would reveal quickly whether this molecule utilizes similar and/or distinct machinery components. At this exploratory stage, either conclusion would be interesting and informative.